1. Field of the Invention
The present invention in the field of medicine relates to novel compositions and methods for the treatment of wounds and for the promotion of more rapid wound healing with diminished scarring.
2. Description of the Background Art
Despite significant progress in reconstructive surgical techniques, scarring can be an important obstacle in regaining normal function and appearance of healed skin. This is particularly true when pathologic scarring such as keloids or hypertrophic scars of the hands or face causes functional disability or physical deformity. In the severest circumstances, such scarring may precipitate psychosocial distress and a life of economic deprivation.
Healing wounded tissue is among the most essential, dramatic and visible jobs performed by the body. Significant progress has recently been made in understanding the sequence of events occurring when traumatized tissue heals. Several dozen different growth factors, or cytokines, have been identified that participate in healing. These growth factors signal the blood to coagulate and plug the gap, they attract immune cells to fight infiltrating microorganisms, and ultimately promote neighboring skin cells to cover the wound. If the wound is sufficiently large, these factors stimulate production of new skin, new blood vessels, new supporting connective tissue and even new bone.
Adult Wound Healing
Adult wound healing in response to injury results in restoration of tissue continuity (Adzick N. S. et al. (eds), FETAL WOUND HEALING, Elsevier, New York 1992, Chapters 1-3, 12, 13 and references cited therein). While some amphibians heal by regeneration, adult mammalian tissue repair involves a complex series of biochemical events that ultimately ends in scar formation. The events occurring during wound repair resemble the process of development, including synthesis, degradation and resynthesis of the extracellular matrix (ECM) (Smith L. T. et al. (1982) J Invest Dermatol 79:935-1045; Blanck C. E. et al. (1987) J Cell Biol 105:139(A)). The ECM contains several macromolecules, including collagen, fibronectin, fibrin, proteoglycans, and elastin (Cohen J. K. et al. (1983) BIOCHEMISTRY AND PHYSIOLOGY OF THE SKIN. New York: Oxford University Press, pp 462-470, 1983; Alvarez O. M., In: CONNECTIVE TISSUE DISEASE: MOLECULAR PATHOLOGY OF THE EXTRACELLULAR MATRIX, Uitto J. et al., eds, New York: Marcell Decker, pp. 367-384, 1986; Murphy-Ullrich J. E. et al., supra, at pp. 455-473). When the injury involves the dermis, repair also entails the removal of cellular debris (Grinnel F. et al. (1981) J Invest Dermatol 76:181-189) and the laying down of a new ECM over which epidermal continuity can be reestablished. This process of repair and dermal matrix reorganization is manifested as scar formation and maturation.
Microscopically, the scar can be identified by its abnormal organization of cellular and matrix elements when compared to surrounding uninjured skin. Grossly, normal scars progress towards stability and maturity. An immature scar is raised, red, and firm, whereas a mature scar is flat, white, and soft. However, not all healing follows this pattern and can result in abnormal scars, such as hypertrophic or keloid scars. Both of these types of scars can be differentiated clinically and histomorphologically from normal scar, but this invariably involves repeated observation over a period of time, as hypertrophic scars in particular can progress to the maturity of a normal scar albeit over a much longer time course.
Adult wound repair includes the stages of hemostasis, inflammation, proliferation, and remodeling. Hemostasis includes vasoconstriction, platelet aggregation and degranulation, blood clotting, and fibrin formation. Inflammation represents a cellular cascade beginning with polymorphonuclear leukocytes (PMNs) followed by macrophages and lymphocytes. This stage also provides host defenses against bacterial infection and contributes numerous growth factors, cytokines, and extracellular matrix (ECM) components. The wound macrophage is the crucial inflammatory effector cell that coordinates adult wound repair (Knighton D. R. et al. (1989) Prog Clin Biol Res 299:217-226).
The proliferative stage involves multiplication of fibroblasts and endothelial and epithelial cells. The initial proteoglycan-rich fibrin matrix is replaced by collagen. In the final remodeling stage, collagen is cross-linked to form a mature scar. In abnormal wound healing conditions such as keloids, hypertrophic scars, strictures, and intraabdominal adhesions, the final result of wound repair creates a cosmetic or functional problem.
Based on the fact that scar formation and maturation involves a complex interaction of dermal and epidermal cells with the ECM, an artificial ECM model has been used to guide the laying down of a new ECM which results in less scarring (Yannis I. V. et al. (1989) Proc Natl Acad Sci U.S.A. 86:933-937). Tension can influence the orientation of organizing collagen, based both on clinical observations and in vitro studies of contracting collagen matrices (Burd D. et al. (1989) Proc Amer Burns Assoc, p. 54).
Growth Factors and Wound Healing
Manipulation of the wound healing environment by the application of extrinsic growth factors such as fibroblast growth factor (FGF) and transforming growth factor-.beta. (TGF.beta.) (Mustoe T. A. et al. (1987) Science 237:1333-1336; Seyedin S. M. et al. (1986) J Biol Chem 261:5693-5695) can influence the early stages of scar formation. The term "TGF.beta." represents a family of 25 kDa dimeric proteins that influence important cell-cell and cell-matrix interactions during embryogenesis, immune responses, and tissue repair. During tissue repair, TGF.beta. modulates the inflammatory response as a potent chemoattractant for fibroblasts, macrophages, neutrophils and T lymphocytes. TGF.beta.1 promotes ECM accumulation by increasing the transcription of genes for collagen, fibronectin and glycosaminoglycans and by inhibiting the breakdown of these macromolecules (as described herein). TGF.beta. can also up-regulate cell surface expression of the integrins that act as receptors for fibronectin, collagen, laminin, and vitronectin thereby influencing cell adhesion and migration. TGF.beta. enhances the epithelial covering of exposed dermis and increases tensile strength in incisional wounds.
Three mammalian isoforms of TGF.beta. are known which exhibit an 80% amino acid sequence homology. Until recently, the TGF.beta. isoforms were thought to be functionally identical, although more recent demonstration of different in vivo effects compared to in vitro activity, and knowledge of the distinction between the three isoforms has prompted further analysis. Immunohistochemical analysis using anti-peptide antibodies specific for each TGF.beta. isoform has shown distinct expression patterns for each isoform in embryogenesis and carcinogenesis. Distinct promoters for the human TGF.beta.1, TGF.beta.2, and TGF.beta.3 genes provides a mechanism for the observed differential expression in selected tissues. This data coupled with the fact that the three isoforms are 98% conserved across species implies both specific function and complex gene regulation for each TGF.beta. isoform in vivo, reinforcing the notion that the three isoforms are not simply interchangeable (Seyedin et al., supra). During repair, specific roles for TGF.beta. isoforms are poorly understood.
Fetal Wound Healing
Human fetal surgery has been successfully performed to treat life-threatening fetal urinary tract obstruction and diaphragmatic hernias (Harrison M. R. et al. (1982) N Engl J Med 306:591-593; Harrison M. R. et al. (1987) J Pediatr Surg 22:556-558). Following the successful delivery of such babies, it has been observed that scarring or contracture around the decompressing hydronephrostomy tubes was absent. Numerous studies have shown that fetal wounds heal without scarring (Adzick N. S. et al. (1985) J Ped Surg 20:315-319; Siebert J. W. et al. (1990) Plast Reconstr Surg 85:495-502). Immunohistochemical and biochemical studies (Longaker M. T. et al. (1990) J Ped Surg 25:63-69; Adzick et al., supra; Burd D. et al. (1990) Brit J Plast Surg 43:571-577) indicate that, as in adults, fetal skin wounds also possess a repair matrix which includes collagen. However in contrast to adult healing, the matrix is rapidly and efficiently organized to appear scarless.
The present invention is intended to exploit knowledge gained from work on fetal wound healing and describe the sequencing of a putative fetal protein factor involved in collagen and matrix organization.
Environmental Differences
Numerous intrinsic and extrinsic differences between the fetus and the adult may drastically influence wound repair. Fetal skin wounds are continually bathed in warm, sterile amniotic fluid rich in growth factors that are crucial to fetal development (Azdick et al., supra). Amniotic fluid is also a rich source for ECM components such as hyaluronan (HA) and fibronectin. Amniotic fluid could modulate fetal skin wound repair simply by supplying HA and fibronectin directly onto fetal skin wounds and by providing growth factors to simulate fetal wound cells to make a unique wound matrix (Azdick et al., supra).
To investigate the influence of the fetal environment on adult tissue repair, full-thickness sheep skin was transplanted onto the backs of 60-day fetal lambs (term=145 days) (Azdick et al., supra), which at that age do not reject allogeneic skin grafts. The adult skin graft was thus bathed in amniotic fluid and perfused by fetal blood; 40 days later (at 100 days gestation), incisional wounds were made on both the adult skin grafts and adjacent fetal skin, and immunohistochemical analysis was performed 7 and 14 days post-wounding. By 14 days the fetal wounds had healed without scarring, while the adult wound collagen pattern was in a typical scar pattern. Thus, neither the amniotic fluid environment nor perfusion by fetal blood prevented scar formation in the wounded adult skin graft. This suggested that the ability of fetal skin to heal without scar formation may be a function of the fetal cells and matrix with or without a fetal environmental influence.
Intrinsic environmental differences include fetal tissue oxygenation, as the fetus depends on transplacental transport from the maternal circulation to meet its oxygen requirements. Because there is a large transplacental oxygen gradient between maternal arterial and umbilical venous blood, fetal arterial blood has a very low pO.sub.2 of 20 torr, which is lower than a maskless mountaineer on top of Mt. Everest (Azdick et al., supra). Fetal wound healing in the face of low fetal arterial pO.sub.2 seems paradoxical. The answer may lie in an inherent difference between the responsiveness of fetal and adult fibroblasts to differing levels of hypoxemia (Longaker M. T. et al. (1993) Plast Surg Res Council).
Some of the properties of fetal skin wound healing may reflect the development of fetal skin. However, healing of fetal bone is also different from adult bone. Virtually no callus formation is present at any time during the healing of fetal lamb bone, and healed fracture sites are indistinguishable radiologically and histologically from uninjured bone. In addition, large bony defects in the fetus, which would be unhealable in infants or adults do close. Not all fetal tissues appear to share the remarkable regenerative qualities of fetal skin and bone. In in utero repair of previously surgically created fetal diaphragmatic hernias, the fetal intestine was always densely adherent to the diaphragmatic defect, but no scar was evident on the previously made thoracic skin incision. Clinical experience with human fetal surgery has shown extensive intraabdominal adhesions following fetal diaphragmatic hernia repair. Thus, fetal mesothelial wounds may heal differently from fetal skin wounds. In addition, amniotic fluid exposure may play an important role in the scarless healing of fetal skin wounds, but its effect on the healing of fetal mesothelial wounds has not been demonstrated.
Fetal Inflammation
Another intrinsic difference between the fetus and adult lies in the inflammatory and immune systems. Histologically, there are few, if any, PMNs in fetal wounds, and there may be a defect in immature PMN chemotactic ability. Fetal lamb wounds lack the typical inflammatory response seen in adult sheep (Longaker M. T. et al., 1990, supra). Because of the prominent role that inflammation plays in adult tissue repair, the minimal fetal inflammatory response to injury may play a pivotal role in the unique fetal repair process. Introduction of adult acute inflammatory cells into the fetus attracts fetal PMNs to the wound site, but an adult fibrotic type of healing response does not follow. These intriguing findings raise questions regarding what attracts fetal fibroblasts into the wounds, how this differs between fetus and adult, and whether characteristic inflammatory mediators of adult wound healing are absent in fetal wounds.
The wound macrophage is the crucial inflammatory cell orchestrating adult wound healing (Knighton et al., supra). Neutrophils can be eliminated from wound repair without a defect in granulation tissue but macrophages cannot. Macrophages are essential regulatory cells that coordinate matrix debridement and turnover, and secrete mediators of inflammation, angiogenesis, and cell growth (Knighton et al., supra). Fetal rabbit wounds, though lacking in PMNs, have an abundance of macrophages (Adzick N. S. et al., 1985, supra). In addition to regulation through growth factor expression, wound macrophages are involved in matrix turnover through proteinase expression. Their secretion of metalloproteinases (e.g., collagenase) and proteinase inhibitors coordinates the degradation and remodeling of the wound ECM. The observation that fetal lamb incisional wounds appear histologically indistinguishable from unwounded skin within two weeks suggests that fetal wound matrix turnover and repair are rapid and efficient (Longaker M. T. et al., 1990, supra).
Fetal Growth Factors
In the fetus, wounds made before the mid-third trimester heal with a collagen repair matrix so organized as to appear scarless, but as in adults, growth factors can modulate the healing wound.
Addition of TGF.beta. or PDGF converted a fetal injury response to an adult-like injury response (Krummel T. M. et al. (1988) J Ped Surg 23:647-652). Administration of anti-TGF.beta. antibodies blocked the increased fibrosis in a wound treated with TGF.beta.1 (Shah Met al. (1992) Lancet 339:213-214). These results further implicate TGF.beta. in scar formation. In fetal mouse lip wounds that normally heal without scarring, the presence of TGF.beta.1 or .beta.2 isoforms could not be detected immunohistochemically with neutralizing antibodies (Whitby D. J. et al. (1991) Dev Biol 147:207-215). This is in stark contrast to neonatal and adult lip wounds which did immunostain for both isoforms. However, it has been shown that fetal wound fluid is abundant in TGF.beta. even during the period of scarless healing, although, interestingly, there is a change in the relative concentrations of isoforms as gestation progresses (Roberts A. B. et al. (1993) J Cell Biol Supplement 17E).
Thus, the presence of growth factors in vivo in healing wounds demonstrated by immunohistochemical staining, neutralizing antibody techniques, and direct assay of wound chamber fluid, supports the concept that growth factors are important in modulating wound healing in the fetus as in the adult.
Hyaluronan
Hyaluronan (HA), formerly called hyaluronic acid or hyaluronate (Balaz E. A. et al. (1986) Biochem J. 233:903), is found in high concentration in ECM wherever tissue repair occurs after injury (Toole, B. P., In: Hay E. D., ed., CELL BIOLOGY OF THE EXTRACELLULARMATRIX. New York: Plenum Press; pp. 259-294, 1982). HA is a glycosaminoglycan (GAG) laid down early in the matrix of both fetal and adult wounds. Sustained deposition of HA is unique to fetal skin, where injury repair occurs with less scarring and more rapidly than adult injury repair. HA appears to provide an extracellular environment conducive to cell mobility and proliferation that may provide the matrix signal responsible for orchestrating healing by regeneration rather than by scarring in the fetus. The fetal wound matrix is rich in HA (Krummel T. M. et al., 1987, supra; De Palma R. L. et al. (1987) Surg Forum 38:626-628; De Palma R. L. et al. (1989) Matrix 9:224-231)). By implanting PVA sponges into 24 day fetal rabbits or into adult rabbits, it was found that the GAG content of fetal sponges was significantly greater on day 2 through 6 when compared to adult sponges, and had 10 times the amount of GAG found in unwounded fetal skin. The major GAG component was HA, as determined by cellulose acetate electrophoresis followed by alcian blue staining (DePalma et al., 1989, supra; Longaker M. T. et al. (1989) Ann Surg 210:667-672).
A role for HA in the scarless healing in the fetus is supported by studies in which topical application of HA tissue extracts modulated post-natal healing, and, for example, enhanced wound healing in rat tympanic membrane perforations (Hellstrom S. et al. (1987) Acta Otolaryngol 442 (Suppl):7-24). HA facilitated wound healing in diabetic rats by promoting epithelial migration and differentiation (Abatangelo G. et al. (1983) J Surg Res 35:410-416). HA-treated wounds developed a greater early wound breaking strength compared to untreated controls, reportedly due to an early accumulation of oriented collagen fibers (Radelli E. et al. (1982) Int'l. Symp. Cutaneous Development, Aging and Repair, University of Padova, p. 42). However, attributing the wound healing effects exclusively to HA is difficult. It is important to remember that tissue-extracted HA, for example from rooster comb or human umbilical cord, is always "contaminated" with one or more proteins, including collagen (Swann D. A. et al. (1975) Ann Rheum Dis 34 (Suppl):98-100).
The present inventors and their colleagues identified a heterogenous group of HA-protein complexes in normal skin and post-burn scar and confirmed the association of HA and collagen. Further, they found that HA extracted from normal skin, normal scar, and hypertrophic scar demonstrated qualitative and quantitative variation in other non-collagen associated proteins despite identical extraction and purification techniques (Burd D. A. R. et al. (1989) Matrix 9:322-327).